Amyloid ␤ protein (A␤), a pathogenic molecule associated with Alzheimer's disease, is produced by ␥-secretase, which cleaves the ␤-carboxyl terminal fragment (␤CTF) of ␤-amyloid precursor protein in the middle of its transmembrane domain. How the cleavage proceeds within the membrane has long been enigmatic. We hypothesized previously that ␤CTF is cleaved first at the membranecytoplasm boundary, producing two long A␤s, A␤ 48 and A␤ 49 , which are processed further by releasing three residues at each step to produce A␤ 42 and A␤ 40 , respectively. To test this hypothesis, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) to quantify the specific tripeptides that are postulated to be released. Using CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxyl-1-propanesulfonate)-reconstituted ␥-secretase system, we confirmed that A␤ 49 is converted to A␤ 43/40 by successively releasing two or three tripeptides and that A␤ 48 is converted to A␤ 42/38 by successively releasing two tripeptides or these plus an additional tetrapeptide. Most unexpectedly, LC-MS/MS quantification revealed an induction period, 3-4 min, in the generation of peptides. When extrapolated, each time line for each tripeptide appears to intercept the same point on the x-axis. According to numerical simulation based on the successive reaction kinetics, the induction period exists. These results strongly suggest that A␤ is generated through the stepwise processing of ␤CTF by ␥-secretase.

Label-free optical imaging is valuable in biology and medicine with its non-destructive property and reduced optical and chemical damages. Quantitative phase (QPI) and molecular vibrational imaging (MVI) are the two most successful label-free methods, providing morphology and biochemistry, respectively, that have pioneered numerous applications along their independent technological maturity over the past few decades. However, the distinct label-free contrasts are inherently complementary and difficult to integrate due to the use of different light-matter interactions. Here, we present a unified imaging scheme that realizes simultaneous and in-situ acquisition of MV-fingerprint contrasts of single cells in the framework of QPI utilizing the mid-infrared photothermal effect. The fully label-free and robust integration of subcellular morphology and biochemistry would have important implications, especially for studying complex and fragile biological phenomena such as drug delivery, cellular diseases and stem cell development, where long-time observation of unperturbed cells are needed under low phototoxicity.

Huntington disease and its related autosomal-dominant polyglutamine (pQ) neurodegenerative diseases are characterized by intraneuronal accumulation of protein aggregates. Studies on protein aggregates have revealed the importance of the ubiquitin-proteasome system as the front line of protein quality control (PQC) machinery against aberrant proteins. Recently, we have shown that the autophagy-lysosomal system is also involved in cytoplasmic aggregate degradation, but the nucleus lacked this activity. Consequently, the nucleus relies entirely on the ubiquitin-proteasome system for PQC. According to previous studies, nuclear aggregates possess a higher cellular toxicity than do their cytoplasmic counterparts, however degradation kinetics of nuclear aggregates have been poorly understood. Here we show that nuclear ubiquitin ligases San1p and UHRF-2 each enhance nuclear pQ aggregate degradation and rescued pQ-induced cytotoxicity in cultured cells and primary neurons. Moreover, UHRF-2 is associated with nuclear inclusion bodies in vitro and in vivo. Our data suggest that UHRF-2 is an essential molecule for nuclear pQ degradation as a component of nuclear PQC machinery in mammalian cells. Huntington disease (HD)2 and related polyglutamine (pQ) diseases are caused by the expansion of trinucleotide repeats encoding pQ within the mutant gene product (1). The pQ length dependence of disease onset and severity in pQ diseases correlates strongly with the tendency of expanded pQ proteins to aggregate in disease models (2, 3). Because of this pQ chain, the gene product assembles into oligomers, further aggregates to form microscopically visible inclusion bodies (IBs), and shows pathological extensity in diseased brains (4).Aggregated forms of pQ-expanded huntingtin (Htt) can disrupt cellular function in a variety of ways, including inactivation of transcription factors (5, 6) and impairment of the ubiquitin proteasome system (UPS) (7). The precise mechanism of UPS inhibition remains to be solved, but could be of particular interest, because it has been shown to occur in in vivo disease models (8). UPS impairment triggers aggresome formation (9), an active cellular mechanism to enrich cytoplasmic aggregates and autophagy-lysosomal components to the microtubule-organizing center by retrograde transport, which in turn enhances the efficiency and selectivity of autophagic degradation of cytoplasmic aggregates supporting the UPS as an alternative protein quality control (PQC) system (10, 11). However, autophagy is ineffective in clearing nuclear aggregates (12), and there is no known nuclear PQC mechanism other than the UPS. Therefore, the nucleus is a relatively protected environment for aggregates than the cytoplasm.The majority of the pQ proteins are functional in the nucleus, and the strong correlation between aggregate toxicity and nuclear translocation of pQ proteins has led to the hypothesis that the nucleus is the primary center for action of these proteotoxins (13). This was shown by redirecting nuclear ataxin-1 or andro...

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We investigated why the cerebrospinal fluid (CSF) concentrations of Aβ42 are lower in mild cognitive impairment (MCI) and Alzheimer's disease (AD) patients. Because Aβ38/42 and Aβ40/43 are distinct product/precursor pairs, these four species in the CSF together should faithfully reflect the status of brain γ-secretase activity, and were quantified by specific enzyme-linked immunosorbent assays in the CSF from controls and MCI/AD patients. Decreases in the levels of the precursors, Aβ42 and 43, in MCI/AD CSF tended to accompany increases in the levels of the products, Aβ38 and 40, respectively. The ratios Aβ40/43 versus Aβ38/42 in CSF (each representing cleavage efficiency of Aβ43 or Aβ42) were largely proportional to each other but generally higher in MCI/AD patients compared to control subjects. These data suggest that γ-secretase activity in MCI/AD patients is enhanced at the conversion of Aβ43 and 42 to Aβ40 and 38, respectively. Consequently, we measured the in vitro activity of raft-associated γ-secretase isolated from control as well as MCI/AD brains and found the same, significant alterations in the γ-secretase activity in MCI/AD brains.